To explore the function of the MeGLYI-13 gene in iron stress, 40-day-old SC8 cassava seedlings were treated with 450 μmol/L of FeCl3 to induce excessive iron stress. Meanwhile, 40-day-old SC8 cassava seedlings without treatment were used as the control. Root, shoot, and leaf samples were harvested at 0, 2, 6, 12, and 24 h after the iron treatment, separately. In this experiment, two plants and three biological replicates were set for each treatment. Then, all of the samples were frozen by liquid nitrogen for isolating the total RNA using a Plant Total RNA Isolation Kit Plus (Foregene). The MonScript™ RTIII Super Mix with dsDNase (Two-Step) Kit was used to remove the remaining DNA from the RNA and then for reverse transcription of the RNA into cDNA. The qRT-PCR data were reacted by SYBR® Premix Ex TaqTM II reagent (Takara) and detected by the ABI 7900 HT Fast Real-Time PCR System. The relative expression levels of MeGLYI-13 at different times after treatment were calculated and analyzed by the popular 2−ΔΔCT method. The primers used in this experiment are listed in Table S3 .
Abi 7900 ht fast real time pcr system
Manufactured by Takara Bio
The ABI 7900 HT Fast Real-Time PCR System is a high-throughput, automated instrument designed for quantitative real-time PCR analysis. It provides accurate and sensitive detection of target sequences in a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Sybr premix ex taqtm 2 reagent, by Takara Bio (1 mentions)
Plant total rna isolation kit plus, by Foregene (1 mentions)
Primescript rt pcr kit, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Bulge loop mirna qpcr primer set, by RiboBio (1 mentions)
Cdna reverse transcription kit, by Takara Bio (1 mentions)
Abi 7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol, by Takara Bio (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
3 protocols using abi 7900 ht fast real time pcr system
1
Investigating MeGLYI-13 Gene's Role in Cassava Iron Stress
2
RNA Extraction and Real-time PCR Analysis
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RNAs were extracted from cultured cells or tissues using TRIzol (Invitrogen) according to the manufacturer’s instruction. cDNAs were reverse transcribed from 0.5 µg total RNAs by PrimeScript RT-PCR kit (Takara Bio Inc.). Real-time PCR was performed with PrimeScript RT reagent kit (Takara Bio Inc.) on an ABI 7900HT Fast Real-time PCR System. Relative expression levels of target genes were quantitatively normalized against the expression of GAPDH using the ΔΔCT method. All real-time PCR primers used in this study are described in Table S2.
3
Quantitative Analysis of MCC and miR-4261 Expression
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Total RNA was extracted from tissues or cells using TRIzol (TaKaRa) according to the manufacturer’s instructions. The final concentration of RNA samples was determined by NanoDrop mass spectrometry (Thermo). For MCC mRNA analysis, first-strand cDNA was obtained using the cDNA reverse transcription kit (TaKaRa), and the SYBR (TaKaRa) was used for qPCR on the ABI 7900HT Fast Real-Time PCR System (Applied Biosystems). GAPDH was used as internal control. The primer sequences for MCC and GAPDH were as follows: MCC: forward 5′-AATAAACGTCTCCAGCAAACAGA-3′, reverse 5′-CGTTCCTCATAGCGAAGTGTC-3′; GAPDH: forward 5′-ATGACATCAAGAAGGTGGTG-3′, reverse 5′-CATACCAGGAAATG AGCTTG-3′. For miRNA analysis, total RNA was reverse transcribed to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). The expression level of miR-4261 was determined using the Bulge-Loop miRNA qPCR Primer Set (RiboBio) with SYBR (TaKaRa) on ABI 7900HT Fast Real-Time PCR System. 5 s was used as internal control.
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